Hydrogen sulfide and polysulfides induce GABA/glutamate/d-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-d-aspartate (NMDA) receptor activity. However, the presynaptic action of H2S/H2Sn on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H2S/H2S2 applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and d-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H2S/H2S2, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H2S/H2Sn, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.


Results
Establishment of 3MST-knockout (KO), TRPA1-KO, and 3MST/TRPA1 double KO rats and their tissue morphology 3MST-KO and TRPA1-KO rats were developed to examine the effect of 3MST and TRPA1 channels on the release of neurotransmitters, the induction of LTP, and the behavior.3MST/TRPA1-double-KO rats were obtained by mating 3MST-KO with TRPA1-KO rats until both alleles of 3MST and TRPA1 were mutated (Supplementary Fig. 1).Using CRSPR-Cas9 technology, we inserted sequences into or deleted them from exon 2 of the 3MST gene for 3MST-KO production, while only inserting sequences into exon 1 of the TRPA1 gene to generate TRPA1-KO (Fig. 1a).These insertions or deletions caused a frameshift in the coding regions of the 3MST and TRPA1 genes.We mated 3MST-KO rats with the #5 construct and TRPA1-KO rats with #8 for the present study until both alleles of each gene were mutated (Fig. 1a; Supplementary Fig. 1).
Western blot analysis with an antibody against 3MST showed that the brains of wild-type rats and TRPA1-KO had a band corresponding to 3MST, while those of 3MST-KO and 3MST/TRPA1 double-KO rats did not (Fig. 1b, Supplementary Fig. 2a-h).The level of 3MST in TRPA1-KO rats was lower than that in wild-type rats.The rhodanese or thiosulfate sulfurtransferase (TST) levels increased in previously reported 3MST-KO mice, in which the transcriptional regulation regions of both genes overlapped 43 .In contrast, in the present study, TST levels did not change in 3MST-KO rats whose control regions were separated (Fig. 1c, supplementary Fig. 2i-p).
It is challenging to detect TRPA1 channels by western blot analysis because of the lack of an antibody specific and sensitive enough to detect channels in the brain, as reported previously 44 .
Morphological differences were examined in the CA1, CA3, and dentate gyrus (DG) of the hippocampus, cerebellum, cerebral cortex, lung, and kidney.No specific morphological differences were observed between tissues (Fig. 1d and Supplementary Fig. 3).

The endogenous levels of H 2 S, H 2 S n , cysteine, and glutathione in the hippocampus
Endogenous levels of cysteine and glutathione in the hippocampus were measured using HPLC, and those of H 2 S, H 2 S 2 , H 2 S 3 , and cysteine persulfide were detected using LC-MS/MS (Fig. 2a-f, Supplementary Fig. 4a,b).The reaction of monobromobimane (MBB) with thiols was performed under alkaline conditions (pH 9.5), which increased levels of H 2 S while decreasing polysulfides 45 .In the present study, an improved method was used in which the reaction of MBB with thiols was performed at physiological pH 7.0 12 .This method enabled us to confirm cysteine and glutathione levels using HPLC.
The levels of cysteine in TRPA1-KO were approximately 60.0% of those in the wild-type, whereas those in 3MST-KO and 3MST/TRPA1-double-KO were not significantly different from those in the wild-type (Fig. 2a).A similar result has been reported in a defective mutant of TRPA1 channels in Drosophila, in which the level of cysteine is 0.50-fold that in the wild-type 46 .The amount of glutathione was not significantly different between KOs and the wild type (Fig. 2b).
The levels of H 2 S were lower in all three KO hippocampi than in the wild type (Fig. 2c), and those of H 2 S 2 were the lowest in 3MST-KO rats, approximately half of those in the wild type (Fig. 2d).TRPA1-KO and 3MST/ TRPA1-double-KO rats also showed lower levels of H 2 S 2 .The 3MST-KO and 3MST/TRPA1-double KO rats showed lower levels of H 2 S 3 than wild-type rats, and all three KO rats had lower levels of cysteine persulfide (Fig. 2e,f).

Endogenous levels of neuro-and glio-transmitters
The endogenous levels of GABA, glutamine, glutamate, d-serine, l-serine, and glycine in the hippocampus were measured by HPLC.GABA levels were significantly higher in TRPA1-KO and 3MST/TRPA1-double-KO rats than in wild-type and 3MST-KO rats (Fig. 2i).This result agrees well with the observation that GABA levels are 1.44 times greater in TRPA1 defective mutant of Drosophila mutants than in the wild-type 46 .The levels of glutamine, which is an essential precursor for neurotransmitters glutamate and GABA, were greater in all three types of KOs than those in the wild-type, while those of glutamate were slightly higher in 3MST-KO and TRPA1-KO but not statistically significant (Fig. 2j,k).
Endogenous d-serine, l-serine, and glycine levels were not significantly different between the KO and wildtype rats (Fig. 2l-n).

H 2 S and H 2 S n induced the release of transmitters from suspended brain cells
To examine whether H 2 S, H 2 S 2 , and H 2 S 3 induce the release of neurotransmitters and related amino acids from brain cells, 20 μM each of Na 2 S, Na 2 S 2 , and Na 2 S 3 , sodium salts of H 2 S, H 2 S 2 , and H 2 S 3 , respectively, were applied to the brain cell suspension prepared from 7 to 14 day-postnatal KOs and wild-type rats.
GABA was released into the extracellular milieu by applying Na 2 S, Na 2 S 2, and Na 2 S 3 .However, the levels induced by Na 2 S 2 and Na 2 S 3 in TRPA1-KO rats were lower than those in the other KO and wild-type rats (Fig. 3a).In contrast, the intracellular GABA concentration did not change significantly (Fig. 3a).GABA is efficiently released into the extracellular milieu via H 2 S, H 2 S 2, and H 2 S 3 .
The release of glycine induced by H 2 S from cells prepared from TRPA1-KO rats and H 2 S 2 and H 2 S 3 from 3MST-KO rats was significantly increased (Supplementary Fig. 5a).The release of d-serine, l-serine, glutamine, or glutamate was not detected in the presence of H 2 S, H 2 S 2 or H 2 S 3 (Supplementary Fig. 5b-e).
The release of d-serine, l-serine, and glutamine by H 2 S, H 2 S 2, and H 2 S 3 may require interactions between neurons and glia.However, the cells were dispersed in the cell suspension and may not have had sufficient interactions between the cells.Therefore, it is necessary to examine transmitters' release into adult rats' hippocampi in vivo.

H 2 S and H 2 S 2 stimulate the release of transmitters in in-vivo microdialysis of the hippocampus
We examined the release of neurotransmitters and gliotransmitters induced by H 2 S and H 2 S 2 in in-vivo microdialysis in the hippocampi of adult rats.Since the in vitro recovery from microdialysis probes has been reported between 0.1 and 7.6% depending on molecules 50 , we examined it on Na 2 S and Na 2 S 2 .Na 2 S was recovered approximately 0.2-1% and Na 2 S 2 was 0.8-4.8%(Supplementary Table 1).Based on these observations, a minimum of 50 times higher concentration (1 mM) of Na 2 S and Na 2 S 2 than that used for the brain cell suspension (20 μM) was applied.
One mM Na 2 S released GABA, glutamate, and glycine at approximately 3.2-, 1.2, and 1.5 times, respectively, of the baseline, and each release increased in a concentration-dependent manner up to approximately 300, 20, and 4 times, respectively, at 10 mM in wild-type rats (Fig. 3b-d).After the cessation of the Na 2 S application, the release returned to the baseline.During the experiments, no specific behavioral changes were observed in any rat.
Na 2 S 2 also induced the release of GABA, glutamate, and glycine from both wild-type and TRPA1-KO rats but was less efficient than Na 2 S (Fig. 3b-g).
d-serine, l-serine, and glutamine were also released by 1 mM Na 2 S, which was approximately 1.4-, 1.5, and 1.4 times than the baseline, respectively, and 1.6-, 1.9, and 1.2 times, respectively, at 10 mM in wild-type rats (Fig. 4a-c).Similar results were obtained for the effect of Na 2 S 2 with no significant difference between wild-type and TRPA1-KO rats (Fig. 4d-f).
These observations suggest that H 2 S and H 2 S 2 induce the release of d-serine, l-serine, and glutamine less efficiently than that of GABA, glutamate, and glycine.

The regulation of the release of H 2 S and polysulfides by neurotransmitters
We also examined whether neurotransmitters induced the release of H 2 S, H 2 S 2 , H 2 S 3 , and cysteine persulfide in the brain cell suspensions.Serotonin significantly decreased the release of H 2 S to approximately 70% of the control in both the wild-type and 3MST-KO brain cell suspensions (Fig. 5a).Serotonin also decreased the intracellular levels of H 2 S to approximately 90% of the control in the wild-type (Fig. 5g).In comparison, it significantly increased the release of H 2 S 3 to approximately 114% of that control (Fig. 5c).
GABA significantly increased the release of cysteine persulfide and cysteine trisulfide from the wild-type but did not induce their release from those prepared from 3MST-KO rats (Fig. 5e and f).GABA also increased the release of H 2 S, H 2 S 2 , and H 2 S 3, but the increase was not significant (Fig. 5a-c).As the molecular weight of cysteine persulfide was too close to that of dopamine to be differentiated with the LC-MS/MS used in the present study, the effect of dopamine was not examined.
Acetylcholine significantly increased the release of cysteine persulfide and the intracellular concentration of H 2 S in the wild type (Fig. 5e and g).Norepinephrine significantly increased the release of cysteine persulfide in wild-type rats but not in 3MST-KO rats (Fig. 5e).
These results were also examined using in vivo microdialysis.However, the release of H 2 S, H 2 S 2 , H 2 S 3 , or cysteine persulfide was not detected, probably because of the interaction of the dialysis membrane with low levels of released H 2 S and H 2 S n (Supplementary Table 1).

Comparison of responses of neurons to NMDA
The sensitivity of neurons to NMDA and high K + levels was examined in primary cultures of hippocampal neurons using Ca 2+ imaging (Fig. 6a-d, Supplementary Fig. 6a,b).The percentage of neurons with strong responses (higher ΔF/F0 values of responses) to NMDA and 50 mM K + was slightly greater in wild-type rats than in all three types of KO rats (Supplementary Fig. 6a,b).Two-sample Kolmogorov-Smirnov cumulative analysis of NMDA/ KCl showed that 3MST-KO and 3MST/TRPA1-double-KO has the significantly (p < 0.01) larger number of cells with small ratios (0.6-1.0) compared to the wild-type, and a similar observation was obtained for TRPA1-KO at 0.6-1.1 (p < 0.05) (Fig. 6b,c).Student's t-test of the ratio for total cells showed that neurons prepared from the wild-type exhibited slightly but significantly (p < 0.01) greater responses to NMDA than those prepared from the three types of KO (Fig. 6d).

The responses of astrocytes to Na 2 S 3 and glutamate
We previously showed that H 2 S 2 and H 2 S 3 activate TRPA1 channels in astrocytes and dorsal root ganglion cells 18,20 .The sensitivity of astrocytes to H 2 S 3 and glutamate was examined by Ca 2+ imaging (Fig. 6e,f) 18,51 .Astrocytes prepared from the wild-type rats and 3MST-KO rats responded to Na 2 S 3 and glutamate (Fig. 6f).In contrast, astrocytes prepared from TRPA1-KO and 3MST/TRPA1-double-KO responded to Na 2 S 3 in a smaller percentage of cells than those prepared from wild-type and 3MST-KO rats (Fig. 6f).Responses to Na 2 S 3 of astrocytes prepared from TRPA1-KO and 3MST/TRPA1-double KO rats may be mediated by other channels, including TRP Vanilloid 4, which also respond to H 2 S n 23 .

The induction of LTP
Because H 2 S and H 2 S 2 facilitate the release of transmitters (Fig. 3), and because the activation of TRPA1 channels 18,20 by H 2 S 2 and their involvement in the induction of LTP has been proposed 25 , the effect of exogenously applied H 2 S 2 on LTP induction was investigated.The effect of exogenously applied H 2 S 2 on LTP induction in the CA1 area of hippocampal slices was investigated in wild-type rats.The magnitude of LTP induced by high-frequency stimulation was not significantly affected by applying Na 2 S 2 (Fig. 7a,b).This observation suggests that endogenous H 2 S 2 and high-frequency stimulation are sufficient to induce LTP in wild-type hippocampal slices.

LTP in 3MST-KO
To further examine the involvement of H 2 S 2 in LTP induction, the LTP levels in 3MST-KO rats were compared with those in wild-type rats.While applying high-frequency stimulation generated robust LTP in wild-type rats, LTP was significantly reduced in 3MST-KO rats (Fig. 7c,d).However, it recovered to the level induced in the wild type in the presence of 20 μM Na 2 S 2 (Fig. 7c,d), suggesting that H 2 S 2 produced by 3MST is required to facilitate LTP induction.
Because H 2 S and H 2 S 2 induce the release of d-serine, the effect of 5,7-dichlorokynurenic acid (DCKA), a specific inhibitor of the glycine (d-serine)-binding site in NMDA receptors, on the induction of LTP was examined.Even in the presence of 20 μM Na 2 S 2 , LTP was greatly suppressed by DCKA in 3MST-KO rats (Fig. 7c,d).This observation confirms that d-serine plays a role in the induction of LTP downstream of H 2 S 2 .
The involvement of endogenous H 2 S and H 2 S 2 produced by 3MST in basal synaptic transmission and shortterm plasticity was examined by comparing the input-output relationship and paired-pulse facilitation.There was no significant difference in the responses between 3MST-KO and wild-type rats (Supplementary Fig. 7a,b), indicating that basal synaptic transmission in 3MST-KO rats was normal and that the reduced LTP could not be due to differences in basal synaptic efficiency.

LTP in TRPA1-KO
Since H 2 S 2 facilitates LTP induction, the involvement of its target protein, TRPA1 channels, in LTP induction was examined.The magnitude of LTP measured in TRPA1-KO rats was greatly reduced compared to that in wild-type rats (Fig. 7e).In contrast, the application of d-serine recovered the LTP to the level observed in the wild-type strain.These observations agree with previous findings 25 that activating TRPA1 channels is critical for LTP induction (Fig. 7e,f, Supplementary Fig. 7c), although the release of d-serine is not dependent on TRPA1 channels (Fig. 4d).
The basal synaptic transmission in TRPA1-KO rats is normal and that the difference in basal synaptic efficiency cannot cause the reduced LTP (Supplementary Fig. 7d,e).

LTP in 3MST/TRPA1-double-KO
The effect of deficiency in both H 2 S 2 production and TRPA1 channel activity on the induction of LTP was examined in 3MST/TRPA1-double-KO rats and compared with that in wild-type rats.The magnitude of LTP in 3MST/ TRPA1-double-KO was greatly reduced (Fig. 7g).Application of 20 μM Na 2 S 2 did not significantly recover LTP (Fig. 7g,h).In contrast, the application of 10 μM d-serine restored the magnitude of LTP to levels not significantly different from those of the wild-type (Fig. 7g,h), suggesting that d-serine application compensated for the lack of 3MST and TRPA1 channels.
The basal synaptic transmission in 3MST/TRPA1-double-KO was normal and that the difference in basal synaptic efficiency could not cause the reduced LTP (Supplementary Fig. 7f,g).

Hyperlocomotion induced by MK-801 in 3MST-KO rats
Hypersensitivity to NMDA antagonists is associated with the positive symptoms of schizophrenia 52 .We examined the hyperlocomotion induced by the acute administration of MK-801 to 3MST-KO and TRPA1-KO rats, and their responses were compared to those induced in wild-type rats.MK-801 induced hyperlocomotion in all three groups.However, 3MST-KO rats showed a significant increase in locomotor activity after MK-801 administration compared with the other two groups (Fig. 8a,b).Bonferroni's post hoc tests revealed that the distance traveled by 3MST-KO rats was significantly greater than that by the wild-type rats 10 min after MK-801 injection, and this trend persisted until the end of the measurement (Fig. 8a).In contrast, TRPA1-KO rats showed a significant decrease in locomotion 15 min after MK-801 injection (p < 0.05).Wild-type and KO rats showed equivalent locomotor activity 30 min before the injection of MK-801 (Fig. 8b).
The involvement of the 3MST and TRPA1 channels in working memory was examined using the Y-maze.The total arm entry of both 3MST-KO and TRPA1-KO rats was significantly lower than that of wild-type rats (Supplementary Fig. 8b).However, the percentage of spontaneous alternation in 3MST-KO rats showed a decreasing tendency but was not significantly lower than that in wild-type rats (Supplementary Fig. 8a).The other behavioral tests, open field, pre-pulse inhibition (PPI) and contextual fear conditioning tests, did not show any significant differences between the genotypes (Supplementary Fig. 8c-h).
The target molecules of H 2 S and H 2 S 2 for regulating transmitter release are localized to the extracellular side of synapses or glia, where cysteine residues can be in oxidized forms, such as cysteine disulfide bonds and nitrosylated or sulfinated cysteine residues.They can be S-sulfurated by H 2 S more efficiently than with H 2 S 2 .Since the intracellular concentrations of H 2 S are greater than those of H 2 S 2 (Fig. 2c,d), H 2 S may regulate the release of these transmitters to a greater extent than H 2 S 2 .
Since the effect of H 2 S on neurotransmitter release during in vivo microdialysis was investigated using high concentrations of Na 2 S and Na 2 S 2 (Figs. 3, 4), the observed effect of Na 2 S can be induced by the polysulfides produced by the oxidation of Na 2 S and generated by the subsequent interaction of the produced polysulfide 18,53 .However, this is unlikely because the effect of Na 2 S on the release of GABA and glutamate is greater than that of Na 2 S 2 , which can S-sulfurate cysteine residues, similar to other polysulfides (Fig. 3).
The expected concentrations in the target region of in vivo microdialysis in the present study may not be far from the physiological concentrations or at least less than the toxic levels.Exposure of rats to high levels of H 2 S during perinatal development significantly depressed neurotransmitters 27 , and sublethal doses of NaHS applied to rodents suppressed neurotransmitters 28 .These observations suggest that toxic concentrations of H 2 S suppress neurotransmitter levels.
The release of d-serine, l-serine, and glutamine induced by H 2 S and H 2 S 2 was not concentration-dependent and less efficient than that of GABA, glutamate, and glycine (Figs. 3, 4), probably because the release mechanism of one group was different from that of the other.
H 2 S and H 2 S 2 showed similar neuroprotective effects against oxidative stress by increasing the intracellular concentrations of glutathione 54 by activating the cystine/glutamate antiporter and cysteine transporter [54][55][56] .Alanine-serine-cysteine transporter 1 (Asc-1), a neutral amino acid transporter located in the neurons, mediates the transport of d-serine, glycine, and l-serine 57,58 .Another transporter system, Slc38a1, which is localized to GABAergic neurons in the hippocampus, is also involved in the release of d-serine 59 .These transporters are candidate molecules releasing d-serine, l-serine, and glutamine induced by H 2 S and H 2 S 2 (Fig. 9a).
d-serine release is enhanced by glutamine, whereas it is suppressed by glycine 39,[59][60][61] .The release of d-serine was not in a concentration of H 2 S and H 2 S 2 dependent manner, probably due to the suppressive effect of glycine, which is released more readily than glutamine (Figs.3d,g, 4a,b) 60 .
The release of GABA and glutamate by H 2 S and H 2 S 2 must be clarified to determine whether H 2 S/H 2 S 2 excites neurons or stimulates the presynaptic vesicular machinery.Some neurotransmitters regulate the release of H 2 S and polysulfides (Fig. 5), suggesting that they can directly or indirectly activate 3MST.Malfunction of the serotonergic system, which regulates the release of H 2 S, can result in various pathological conditions, including schizophrenia 62 .Understanding the physiological roles of H 2 S and polysulfides both upstream and downstream of these neurotransmitters as well as the regulation of 3MST activity in the nervous system is essential.
Glutamatergic pyramidal neurons and GABAergic interneurons contain SR that release d-serine.GABAergic neurons activate GABA A receptors on astrocytes, which contain higher concentrations of Cl − ions in the cytoplasm to depolarize the membrane 63 , release d-serine 53 , and stimulate nearby neurons to release d-serine.
The present study agrees with a previous study 25 in that LTP requires TRPA1 activity (Fig. 7e,f), suggesting that astrocytes with activated TRPA1 channels may influence the activity of neighboring neurons 64 , leading to LTP induction (Fig. 9a).The sensitivity of NMDA receptors may be altered depending on the age of the animals,  owing to changes in the subunits of the receptors 65 .Therefore, the sensitivity of NMDA observed in neuronal cultures may differ from that observed in adult rats examined for LTP induction.However, because LTP induction was investigated using different genotypes of animals in the same age group, the influence may be small.The present study showed that MK-801 application to 3MST-KO rats significantly increased hyperlocomotion (Fig. 8).NMDA antagonists induce a behavioral phenotype that resembles the symptoms of schizophrenia, including cognitive impairments 52,66 .Chronic inactivation of NMDA receptors during the neonatal period affects behavioral properties later in life.For example, deleting NR1, a subunit of the NMDA receptor, in corticolimbic interneurons in the early postnatal period augments MK-801-induced hyperlocomotion in adulthood 42 .Since the release of H 2 S and H 2 S 2 was decreased in 3MST-KO rats (Fig. 2c,d), the activity of NMDA receptors may be chronically reduced (Fig. 9b).This mechanism may be involved in the increase in hyperactivity induced by the acute application of MK-801 in 3MST-KO rats.
The endogenous levels of H 2 S and polysulfides in TRPA1-KO rats were as low as those in 3MST-KO rats because of secondary effects caused by the low levels of 3MST and cysteine in TRPA1-KO rats (Fig. 2a,c-f).Since cysteine is a substrate of CAT, whose activity is regulated by Ca 2+ to produce 3MP 67 , the activity of TRPA1 channels can regulate cysteine levels.In contrast, cysteine levels in 3MST/TRPA1-double-KO rats were reinstated (Fig. 2a), probably because the accumulated 3MP in the absence of 3MST may induce feedback inhibition of CAT activity.It is necessary to clarify the mechanism of the regulation by TRPA1 channels of the transcription or translation of 3MST as well as the metabolism of cysteine.
The 3MST and TRPA1 channels play critical role in the induction of LTP, whereas only 3MST is involved in behavioral hyperactivity (Figs. 7, 8).TRPA1-KO rats had endogenous levels of H 2 S/H 2 S 2 as low as those of 3MST-KO rats; however, TRPA1-KO rats still had low levels of 3MST (Fig. 1), which can release the newly synthesized H 2 S and polysulfides.These differences may differentiate the behavioral hyperactivity between 3MST-KO and TRPA1-KO rats.
In conclusion, H 2 S and polysulfides regulate the release of several neurotransmitters, some of which control the release of H 2 S and polysulfides.H 2 S/H 2 S 2 produced by 3MST is involved in hyperactivity and, together with TRPA1 channels, regulate LTP induction.These molecules, their enzymes, and target molecules have therapeutic potential in psychiatric diseases and cognitive decline.

Chemicals
All methods were performed in accordance with the guidelines and regulations of chemical substance management and approved by the committees of chemical substance management in the Sanyo-Onoda City University, Musashino University and National Institute of Neuroscience, National Center of Neurology and Psychiatry.Na 2 S 2 (Dojindo, Kumamoto, Japan), Na 2 S 3 (Dojindo), Na 2 S (Wako pure chemicals, Osaka, Japan), l-cysteine (Wako), and glutathione (Wako) were dissolved at 0.1 M in ultrapure water.These stock solutions were stored at − 80 °C, and were used within a week.5,7-Dichlorokynurenic acid (DCKA) was obtained from Tocris Bioscience (Avonmouth, UK).d-Serine and the other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Generation of 3MST-and TRPA1-deficient rats
Each founder rat harboring the 3MST mutant allele in exon 2 or the TRPA1 mutant allele in exon 1 was crossed with wild-type rats to obtain 3MST-or TRPA1-heterozygous rats.The heterozymous rats were crossed to obtain homozygous rats.Genotyping was performed by means of PCR and determined by sequencing.The genotyping primers were 5′-TGG TAT CTT TCC TGT CTT GCAG-3′ and 5′-CGA AAT GCG TGG CAC TAG G-3′ for 3MST, and 5′-CAG AAC CGG CTT TAG CTT CA-3′ and 5′-GCC GTG CTT CCT AAA CTT GA-3′ for TRPA1.

Histology
For histological analysis, male rats were anesthetized with a mixture of medetomidine (0.375 mg/kg), midazolam (2.0 mg/kg), and butorphanol (2.5 mg/kg) and perfused with 0.02 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA).Then the organs (brain, lung, and kidney) were quickly removed and post-fixed in 4% PFA for more than 24 h.These samples were cryoprotected with 30% sucrose PFA, then quickly frozen with dry ice-hexane cooling bath.Samples were cut using microtome (Yamato Kohki, Osaka, Japan) and 8-µm sections were collected.For the brain, sagittal and coronal sections were obtained.These sections were mounted on glass slides and air-dried before staining.Coronal sections of the brain containing the dorsal hippocampus were stained with cresyl violet.For the remaining sections, hematoxylin-eosin (HE) staining was conducted.Photographs of each section were obtained using a microscope (Olympus) equipped a digital camera (Wraymer, Osaka, Japan) connected to a computer.

Preparation of brain tissue for LC-MS/MS and HPLC analysis
For LC-MS/MS analysis of H 2 S n and cysteine persulfide, brain extracts were prepared according to the previously reported method with some modification 12 .Briefly, hippocampus removed from 30 day old male F344 rats, was homogenized with 9 volume of ice-cold homogenizing buffer (10 mM phosphate buffer pH7.0 containing 1% Triton X-100, EDTA-free complete as protease inhibitor, 0.1 mM diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid (DTPA) and 2 mM monobromobimane) and centrifuged at 12,000×g for 10 min at 4 °C.Supernatant was transferred to a new tube and left on bench for 30 min in dark at room temperature to label thiol residues of H 2 S n and cysteine persulfide.Reaction was stopped by adding 5-sulfosalicyclic acid (SSA: final concentration 2%) and incubated for 15 min on ice.The reaction mixture was centrifuged at 12,000×g for 10 min, supernatant was analyzed with LC-MS/MS (Agilent 6470 Triple Quad LC/MS, Santa Clara, USA).
For HPLC analysis of amino acid, brain extracts were prepared according to the previously reported method with minor modification 68 .Briefly, hippocampus was homogenized with 5 volume of methanol at 1500 rpm for 10 strokes, left on bench for 10 min, centrifuged at 10,000×g for 10 min at 4 °C.Supernatant was analyzed with HPLC (Waters 2695, Milford, USA).

Suspensions of brain cells
The suspensions of brain cells were prepared by the modified method reported previously 69 .Briefly, brains of male F344 rats were removed at the postnatal day 7-14.After meninges were removed, brains were chopped to approximately 1 mm cubes with scissors in the basic medium containing 3 mg/ml BSA fraction V (Sigma-Aldrich, St. Louis, MO, USA), 14 mM glucose (Sigma), 1.2 mM MgSO 4 in Ca 2+ free HBSS (Wako pure chemicals, Osaka, Japan).The suspended brain cubes were centrifuged at 100×g, 4 °C for 20 s to remove supernatant, and washed once with the basic medium.The brain cubes were incubated in 10 ml basic medium containing 0.025% trypsin EDTA (Wako) for 15 min at 37 °C, and then 10 ml basic medium containing 6.4 μg/ml DNase I (Sigma-Aldrich), 0.04 mg/ml Soybean Trypsin Inhibitor (SBTI) (Sigma-Aldrich) was added and gently mixed.The supernatant was removed after centrifugation at 100×g for 20 s at room temperature.Two ml basic medium containing 40 μg/ ml DNase I, 0.25 mg/ml SBTI, and 3 mM MgSO 4 was added to the brain cubes and mixed gently up and down with a pipette without making foams for 30 times.After centrifugation at 210×g for 1 min, cells were recovered and washed with 2 ml HBSS with Ca 2+ and Mg 2+ medium (Wako) containing 14 mM glucose (Sigma-Aldrich) for 3 times, and then preincubated at 37 °C for 1 h in a shaker at 100 rpm (Taitec Bio-shaker BR-40LF, Saitama, Japan) before used for experiments.

Measurement of endogenous polysulfides and neurotransmitters and those released from brain cell suspension
After 1 h preincubation, 500 μl suspensions of brain cells were incubated for 15 min at 37 °C in the presence of 200 μM neurotransmitters (Sigma-Aldrich), centrifuged at 1000×g for 1 min to separate supernatant (cell-sup) and pellet.Pellets were homogenized in homogenizing buffer and centrifuged at 12,000×g for 10 min at 4 °C to recover supernatant (pellet-sup).
Thiol residues of Na 2 S n and cysteine persulfides in cell-sup and pellet-sup were labelled with monobromobimane as described above and analyzed by LC-MS/MS.

Measurement of transmitters released and changes in their endogenous levels in cells
After preincubation, brain cell suspensions were incubated for 15 min at 37 °C in the presence of 20 μM Na 2 S, Na 2 S 2 and Na 2 S 3 .Na 2 S n (n = 1-3) were dissolved and diluted in water until 100 times of the final concentrations to prevent the production of S8 precipitates, then dissolved into the corresponding buffer solution.After the exposure to Na 2 S n the suspensions of brain cells were centrifuged to separate supernatant (cell-sup) and pellets.Pellets were homogenized with methanol and centrifuged at 10,000×g for 10 min at 4 °C to separate supernatant (pellet-sup).Cell-sup and pellet-sup were analyzed with HPLC.

HPLC analysis
For HPLC measurement of amino acids including d-serine the method by Grant et al. 70 was employed.Automated pre-column derivatization was carried out by drawing up a 5 µl aliquot of sample solution and 5 µl of derivatizing reagent solution consisting of 1 mg o-phthaldialdehyde and 2 mg N-isobutyryl-l-cysteine in 0.1 ml methanol and 0.9 ml 0.2 M sodium borate buffer (pH 10), then holding in the injection loop 5 min prior to injection to the HPLC (Waters alliance 2695).Amino acids were separated by a Symmetry C18 column (4.6 mm × 150 mm, 3.5 µm).The flow rate was 0.7 ml/min and run time was 85 min.Solvent A comprised 850 ml 0.04 M sodium phosphate and 150 ml methanol (pH 6.2).Solvent B comprised 670 ml 0.04 M sodium phosphate, 555 ml methanol, and 30 ml tetrahydrofuran, adjusted to pH 6.2.The amino acids were separated by a concave gradient from 15 to 100% B in 50 min.The solvent mix was returned to initial conditions by 65 min using a concave gradient and maintained at that composition for 20 min prior to the next injection.Amino acid derivatives were monitored at excitation and emission wavelengths of 260 and 455 nm, respectively (Water 2475 fluorescence detector).

In vivo microdialysis
Prior to the experiments, male F344 rats at 7-8 weeks old were implanted with microdialysis guide cannula targeting the dorsal hippocampus under the same anesthesia as described in Histology.After surgery, animals were allowed to recover for at least 24 h before experiments.At the start of experiments, the dialysis probes (A-I-3-01; EICOM, Kyoto, Japan) with 1.0 mm-long membranes were inserted into the guide cannula and were continuously perfused with perfusion medium (150 mM NaCl, 2.2 mM CaCl 2 , 4.0 mM KCl) at a flux rate of 2.0 µl/min using a syringe pump (ESP-64; EICOM).Each rat was attached to a swivel unit to allow free movement.After 60 min equilibration period, perfusates were collected every 20 min.Following 60 min baseline sampling, Na 2 S 2 or Na 2 S (1 mM, 3 mM, and 10 mM) dissolved in perfusion medium were perfused for 20 min at 40 min intervals in ascending order.After 100 min of additional sampling, rats were sacrificed under isoflurane anesthesia and the location of the dialysis probes was verified in 40 μm-thick brain slices.
High-performance liquid chromatography with an electrochemical detector (HTEC-700, EICOM, EICOM, Kyoto, Japan) was used for quantification of amino acids in micridialysis experiments.For quantification of d-serine, l-serine, glutamate, glutamine, and glycine, an aliquot of each dialysate sample was derivatized with N-acetyl-l-cysteine and o-phthaldialdehyde for 300 s at 10 °C and then analyzed on a reverse-phase column (Eicompak EX-3ODS, 4.6 φ × 100 mm, EICOM) operated at the constant flow rate of 0.5 ml/min at 30 °C.The potential of the glassy carbon electrode (WE-GC, EICOM) was set at + 0.6 V (vs.Ag/AgCl).Composition of the mobile phase for the measurement was 100 mM phosphate buffer (pH 6.0), methanol (18 v/v %) and 5 mg/L EDTA•2Na.For quantification of GABA, an aliquot of each dialysate sample was derivatized with 2-mercaptoethanol and o-phthaldialdehyde for 150 s at 10 °C and then analyzed on a reverse-phase column (Eicompak FA-30DS, 3 φ × 75 mm, EICOM) operated at the constant flow rate of 0.5 ml/min at 40 °C.The potential of the glassy carbon electrode (WE-GC, EICOM) was set at + 0.6 V (vs.Ag/AgCl).Composition of the mobile phase for the measurement was 100 mM phosphate buffer (pH 6.0), methanol (7 v/v %), acetonitrile (13 v/v %) and 5 mg/L EDTA•2Na.

Primary cultures of neurons
Brains were removed from postnatal day 3 to 7 of male F344 rats and the hippocampus was dissected in L15 medium (Life Technologies).The tissue was chopped and digested with 0.25% trypsin (Sigma-Aldrich) and 0.1% DNase I (Sigma-Aldrich) in Ca 2+ /Mg 2+ -free PBS for 15 min at 37 °C.After mechanical dissociation, cells were plated onto poly-d-lysine-coated 35 mm dishes (BD Biosciences, San Jose, CA, USA) and cultured in Neurobasal medium (Life Technologies) supplemented with B27 (Life Technologies) for 2 days at 37 °C in 5% CO 2 .Cells were further cultured in the presence of 5 μM cytosine β-d-arabinofuranoside (AraC, Sigma-Aldrich) for 1 day, and washed once with Neurobasal medium supplemented with B27 to remove AraC.Cells cultured for additional 3-4 days were used for experiments.

Ca 2+ imaging of neurons and astrocytes
The cells were washed once with basal salt solution (BSS) consisting of 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 5.5 mM glucose, and 10 mM HEPES-NaOH (pH 7.3) and were loaded with 1 µM calcium green-1/acetoxymethyl ester (Life Technologies) and 0.01% (v/v) cremophorEL in BSS solution for 50 min at 37 °C.The dishes were mounted on an upright fixed stage microscope (Leica DM-LFS; Leica Microsystems, Wetzlar, Germany) and perfused at 2 ml/min.The images were acquired using a water-immersion objective (X20; 0.5 NA; Leica Microsystems) and a CCD camera (C4742-95-12ER; Hamamatsu Photonics, Shizuoka, Japan).The frame duration ranged from 147 to 207 ms, and each image was acquired at 4 s intervals and 4 X 4 binning.Images were acquired and analyzed using Aquacosmos 2.0 software (Hamamatsu Photonics).Changes in calcium concentrations were monitored as a change in the fluorescence intensity (F) relative to the control image (F0) that was acquired before stimulation.

Field EPSP recordings and induction of LTP
Preparation of hippocampal slices and recording of evoked potentials were made as described in our previous papers 71 .In brief, 400 μm hippocampal slices were prepared from male wild type (F344), 3MST-KO, TRPA1-KO, and 3MST/TRPA1-double KO rats (5-13 weeks old) and maintained in a chamber at 30 °C, where they were continuously perfused with oxygenated (95% O 2 :5% CO 2 ) artificial cerebrospinal fluid (ACSF).The composition of the ACSF was as follows (in mM): 124 NaCl, 1.24 KH 2 PO 4 , 3 KCl, 2.2 CaCl 2 , 1.4 MgSO 4 , 25.0 NaHCO 3 , and 10.0 glucose.Schaffer collaterals were stimulated by a bipolar tungsten electrode placed in the stratum radiatum of the CA1 region near the CA2/CA1 border, and the evoked field EPSPs (fEPSPs) were recorded using an extracellular glass microelectrode filled with 0.9% NaCl (tip resistance 2-8 MΩ) placed in stratum radiatum of the CA1 region.Signals were amplified using Axopatch 200B (Molecular Devices) and digitized with a DigiData 1322A or 1550B (Molecular Devices), and acquired using pClamp software (Molecular Devices).Single-pulse test stimulations (100 μsec duration) were applied at 30-s intervals.The stimulus intensity was adjusted in the range of 25-55 μA to evoke fEPSPs of 50% of the maximum amplitude.To induce LTP, a high-frequency stimulation (100 pulses at 100 Hz) was applied at the same intensity with the test stimulation.The rising slope of fEPSP was measured as an index of synaptic efficacy.

LC-MS/MS analysis
Samples derivatized with monobromobimane (mBB) (Life Technologies) were analyzed by the triple-quadrupole mass spectrometer coupled to HPLC (Agilent technology, LC-MS/MS 6470).Samples were subjected to a reverse phase Symmetry C18 HPLC column (4.6 × 250 mm, Waters) at the flow rate of 1.0 ml/min.The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in methanol.Samples were separated by eluting with a gradient: 5% B at 0-5 min and 5-90% B at 5-25 min.The column oven was maintained at 40 °C.The efuent was subjected to the mass spectrometer using an electrospray ionization (ESI) interface operating in the positive-ion mode.The source temperature was set at 400 °C, and the ion spray voltage was at 4.5 kV.Nitrogen was used as a nebulizer and drying gas.The tandem mass spectrometer was tuned in the multiple reaction

Figure 1 .
Figure 1.Generation of 3MST-KO, TRPA1-KO, and 3MST/TRPA1-double-KO rats, their morphology, the levels of 3MST and TST.a. Schematic representation of rat 3MST and TRPA1 gene structures and sequences of wild-type and mutant alleles.Mutants 3MST #5 and TRPA1 #8 were established and used in the present study.b and c.Western blot analysis of the brains of wild-type, 3MST-KO, TRPA1-KO, and 3MST/TRPA1 double-KO rats, using antibodies against 3MST (b) and rhodanese or thiosulfate sulfurtransferase (TST) (c).(b) and (c) are images cropped from the original blots presented in Supplementary Fig. 2. d.Cresyl violet staining of the hippocampal slices.The CA1, CA3, and dentate gyrus (DG) obtained from 3MST-KO, TRPA1-KO, 3MST/ TRPA1-double-KO, and wild-type rats were stained.

Figure 2 .
Figure 2. The intracellular levels of cysteine, glutathione, H 2 S, H 2 S 2 , H 2 S 3 , cysteine persulfide, bound sulfane sulfur, and transmitters in KO rats compared to those of the wild-type.a and b.The intracellular concentrations of cysteine (a) and glutathione (b) in the hippocampus measured by HPLC.c to f. Intracellular levels of H 2 S (c), H 2 S 2 (d), H 2 S 3 (e), and cysteine persulfide (f) in the hippocampus were measured using LC-MS/MS.g and h.Intracellular levels of bound (sulphane) sulfur in the hippocampus were measured by gas chromatography.i to n. Relative intracellular concentrations of GABA (i), l-glutamine (j), l-glutamate (k), d-serine (l), l-serine (m), and l-glycine (n) in the hippocampus measured by HPLC.W, wild-type; M, 3MST-KO; T, TRPA1-KO; MT, 3MST/TRPA1-double-KO.The experiments were repeated five times.*p < 0.05, **P < 0.01 significantly different as indicated by the Student's t-test.All data are shown mean ± SEM.

Figure 3 .
Figure 3.The release of GABA, glutamate and glycine induced by H 2 S and H 2 S n from the brain cell suspension and in in vivo microdialysis.a.The release of GABA from the brain cell suspension stimulated by 20 μM each of Na 2 S, Na 2 S 2, and Na 2 S 3 and the changes in the intracellular GABA levels.* p < 0.05, **p < 0.01 with Student's t-test.b to g.The release of GABA (b and e), glutamate (c and f), glycine (d and g) induced by H 2 S (b to d) and by H 2 S 2 (e to g) at the concentrations indicated in the figure in the wild-type rats (blue line) and TRPA1-KO (orange line).A baseline is defined as 100.(b), n = 5; (c) and (d) n = 7; (e) the wild-type n = 5, TRPA1-KO n = 8; (f) the wild-type n = 6, TRPA1-KO n = 9; (g) the wild-type n = 9, TRPA1-KO n = 8; All data are shown means ± SEM.Each peak value of the response was compared to the corresponding base line response observed at the start of the application of Na 2 S or Na 2 S 2 .*p < 0.05, **p < 0.01, # p < 0.1 with Student's t-test.

Figure 4 .
Figure 4.The release of d-serine, l-serine and glutamine induced by H 2 S and H 2 S 2 in in vivo microdialysis.a to f.The release of d-serine (a and d), l-serine (b and e), glutamine (c and f) induced by H 2 S (a to c) and by H 2 S 2 (d to f) at the concentrations indicated in the figure in the wild-type rats (blue line) and TRPA1-KO (orange line).A baseline is defined as 100.(a) to (b) n = 7; (d) to (f) the wild-type n = 9, TRPA1-KO n = 8.All data are shown means ± SEM.Each peak value of the response was compared to the corresponding base line response observed at the start of the application of Na 2 S or Na 2 S 2 .*p < 0.05, **p < 0.01, # p < 0.1 with Student's t-test.

Figure 5 .
Figure 5.Serotonin and GABA regulate a release of H 2 S, H 2 S 2 , H 2 S 3, and cysteine persulfide.a to f. Suspended brain cells prepared from the wild-type and 3MST-KO rats were stimulated by 200 μM each of acetylcholine, norepinephrine, GABA, Serotonin (5HT), and glutamate, and released H 2 S (a), H 2 S 2 (b), H 2 S 3 (c), cysteine (d), cysteine persulfide (e), and cysteine trisulfide (f) to the medium were measured.g to i. Suspended brain cells prepared from the wild-type rats were stimulated by 200 μM each of acetylcholine and 5HT, and the intracellular levels of H 2 S (g), H 2 S 2 (h), and H 2 S 3 (i) were measured.The experiments were repeated 8-12 times.* p < 0.05, ** p < 0.01 by student t-test.All data are shown mean ± SEM.

Figure 6 .
Figure 6.Sensitivity to transmitters of neurons and astrocytes prepared from 3MST-KO, TRPA1-KO, and 3MST/TRPA1-double-KO compared to those from the wild-type rats.a and b.Responses of neurons to N-methyl-d-aspartate (NMDA) and high K + measured by Ca 2+ imaging.(a) Typical response to 100 μM NMDA and 50 mM KCl (upper panel) and images of cultured neurons prepared from KO and wild-type rats (lower panel).(b) The ratio of responses to NMDA/ high K + stratified by amplitude (F/F 0 ).c. Kolmogorov-Smirnov cumulative analysis of NMDA/KCl.**p < 0.01, *p < 0.05.d.Student's t-test was used to determine the NMDA/KCl ratio (%) of total cells.WT: 106 ± 0.70, 3MST-KO: 101 ± 0.67, TRPA1-KO: 103 ± 0.86, 3MST/TRPA1-double KO: 100 ± 0.75.**p < 0.01.(e) A typical response of astrocytes to 20 μM Na 2 S 3 and 100 μM glutamate (upper panel) and images of cultured astrocytes prepared from KO and wild-type rats (lower panel).(f) percentage of astrocytes responding to Na 2 S 3 and glutamate (blue), and glutamate alone (orange).The percentage of cells responded to Na 2 S 3 and glutamate in TRPA1-KO and in M/T-KO is significantly different from that in WT cells by the Chi-squared-test, while that of TRPA1-KO is not significantly different from that of M/T-KO.**p < 0.01 and *p < 0.05.Note that cells responded to Na 2 S 3 but not to glutamate were observed 9 out of 423 cells in the wild-type, 5 out of 583 in 3MST-KO, 2 out of 210 in TRPA1-KO, and 0 out of 222 in 3MST/TRPA1-double KO.The experiments were repeated at least ten times.The total cells measured were 173-331 (a-d) and 210-583 (f).

Figure 9 .
Figure 9. Schematic representation of the possible mechanisms of H 2 S and H 2 S 2 in LTP and behavioral hyperactivity.a.The possible mechanisms of H 2 S and H 2 S 2 in LTP.H 2 S and H 2 S 2 produced by 3MST facilitate the release of glutamate and GABA.H 2 S 2 activates TRPA1 channels to induce Ca 2+ influx in astrocytes which excite nearby neurons to induce LTP.Glutamine released from astrocytes enhances the release of d-serine.Glutamate and d-serine activate NMDA receptors to induce LTP.b.The possible mechanisms for the behavioral hyperactivity induced in 3MST-KO rats.The chronic inactivation of NMDA receptors during the neonatal period by applying MK-801 affects adult behavioral properties.The release of glutamate and d-serine enhanced by H 2 S and H 2 S 2 is decreased in 3MST-KO, suppressing the activity of NMDA receptors.Suppression of NMDA receptor activity induces hyperlocomotion in 3MST-KO, similar to the animals treated as the antagonist of NMDA receptors in neonatal periods.